Transcriptomic signatures of individual cell types in cerebral cavernous malformation

Cerebral cavernous malformation (CCM) is a hemorrhagic neurovascular disease with no currently available therapeutics. Prior evidence suggests that different cell types may play a role in CCM pathogenesis. The contribution of each cell type to the dysfunctional cellular crosstalk remains unclear. Herein, RNA-seq was performed on fluorescence-activated cell sorted endothelial cells (ECs), pericytes, and neuroglia from CCM lesions and non-lesional brain tissue controls. Differentially Expressed Gene (DEG), pathway and Ligand-Receptor (LR) analyses were performed to characterize the dysfunctional genes of respective cell types within CCMs. Common DEGs among all three cell types were related to inflammation and endothelial-to-mesenchymal transition (EndMT). DEG and pathway analyses supported a role of lesional ECs in dysregulated angiogenesis and increased permeability. VEGFA was particularly upregulated in pericytes. Further pathway and LR analyses identified vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 signaling in lesional ECs and pericytes that would result in increased angiogenesis. Moreover, lesional pericytes and neuroglia predominantly showed DEGs and pathways mediating the immune response. Further analyses of cell specific gene alterations in CCM endorsed potential contribution to EndMT, coagulation, and a hypoxic microenvironment. Taken together, these findings motivate mechanistic hypotheses regarding non-endothelial contributions to lesion pathobiology and may lead to novel therapeutic targets. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-023-01301-2.


Supplemental Methods Sporadic and Familial diagnosis and genotyping
Familial CCM patients harbor multiple lesions throughout the brain on the most sensitive susceptibility weighted imaging (SWI) MRI sequence, a documented CCM1, CCM2, or CCM3 germline mutation, and/or first-degree relative with a history of CCM.Familial case without genetic testing (e.g., deidentified sample from tissue biobank) was classified as multifocal unknown genotype [1].
Sporadic CCM cases harbored a solitary lesion on SWI MRI sequence, or a cluster of lesions associated with a developmental venous anomaly [1].

Validation of cell population from fluorescent cell activated sorting
A list of gene markers showing over 70% of sensitivity in identifying a cell population was first selected from PanglaoDB to validate fluorescent cell activated sorting (FACS) results (Table S1) [2].Few specific genes markers have been identified in pericytes, therefore CSPG4 (NG2), a well-known marker for pericytes, was also included [3,4].The selected genes for each cell population were then confirmed to be cell-type specific using CellMarker 2.0 [5].The genes were queried within the differential RNA seq profiling of each cell types against all the others regardless the tissue type (all: p<0.1, false discovery rate [FDR] corrected; with absolute fold change [|FC|]>1.5).The putative cell population were considered to be validated if the cell-specific gene expression markers were upregulated, when compared with all the other cell types.

The expression of cell type-related genes is altered in CCMs
The results of the secondary analyses on FC magnitude identified 111 genes between lesional ECs and pericytes (Fig. 1 and Table S4).Of these 111 genes, 19 were previously identified as dysregulated in lesional ECs, and 80 in lesional pericytes (i.e., compared to their respective non-lesional control).
The comparison between ECs and neuroglia identified 42 genes (Fig. 1 and Table S4), including 8 overlapped with DEGs in the lesional ECs, and 31 in lesional neuroglia (i.e., compared to their respective non-lesional control).Finally, the secondary analyses on FC magnitude identified 66 genes between lesional pericytes and neuroglia (Fig. 1 and Table S4).Among these 66 genes, 22 were already defined as dysregulated in lesional pericytes and 39 in neuroglia (i.e., compared to their respective non-lesional control).

Enriched Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways support Ingenuity Pathway Analysis and Hallmark gene set results
Forty-five GO terms and 11 KEGG pathways (p<0.01,FDR corrected) were identified using the 90 common DEGs among all the cell type (Tables S3d, e).These data implied the microenvironment of CCM lesion including inflammation and coagulation and it might lead to endothelial-to-mesenchymal transition in lesion development.
Eighty-one GO terms and 26 KEGG pathways (p<0.01,FDR corrected) were found in lesional ECs, which indicated proliferation of ECs, as well as its involvement with focal and cell adhesion, interactions with extracellular matrix component, and coagulation (Tables S7a and S8a).There were 226 GO terms and 30 KEGG pathways (p<0.01,FDR corrected) identified in CCM pericyte.There results suggested the pericytes' role of antigen processing and presentation, focal and cell adhesion, and endoplasmic reticulum stress in CCM pathogenesis (Tables S7b and S8b).Five hundred sixty-seven GO terms and 112 KEGG pathways (p<0.01,FDR corrected) were identified in neuroglia within CCM lesion.These imply a neuroglia-mediated inflammatory response in CCM disease (Tables S7c and S8c).

Fig. S1
Fig. S1 Endothelial cells, pericytes and neuroglia were sorted and validated in all CCM lesions and non-lesional control brain tissues.(A) Gating of sorted cell population for RNA sequencing.(B), (C),and (D) Selected gene markers of endothelial cells, pericytes and neuroglia were queried in sequencing data.The statistical significance of gene markers is p<0.1,FDR corrected; with |FC| > 1.5.

Fig. S2
Fig. S2 Ligand-Receptor (LR) analysis revealed cell-cell communications via NOTCH signaling among individual cell types in CCM lesion compared to non-lesional control brain.(A) Contribution of each LR interaction of the overall NOTCH signaling in CCM lesion.(B) Contribution of each LR interaction of the overall NOTCH signaling in nonlesional control brain.The color bars of the inner semicircles indicate the target cell type of the outgoing signal (receptor).Ligand families depicted are Delta-Like ligands (DLL) and Jagged (JAG).The significant results of LR interaction were defined as p<0.05,FDR corrected.The list of LR interactions among all the cell types is available in Supplemental Table 9.